We have investigated the mechanism of post-Golgi transport and delivery of hormone and BDNF vesicles to the plasma membrane for activity-dependent secretion which is critical for endocrine function and synaptic plasticity. We showed that the CPE cytoplasmic tail on these secretory vesicles plays an important role in their transport. Overexpression of the CPE cytoplasmic tail in the cytoplasm to compete with the endogenous tail diminished localization of endogenous POMC, BDNF and fluorescence-tagged CPE in the processes of an endocrine cell line, AtT20;and hippocampal neurons. In live hippocampal neurons, primary pituitary and AtT20 cell images, overexpression of the CPE tail inhibited the movement of BDNF- and POMC/CPE-containing vesicles to the processes, respectively. S-tagged CPE tail pulled down endogenous microtubule-based motors, dynactin (p150), dynein and KIF1A/KIF3A from cytosol of AtT20 and brain cells. Finally, overexpression of the CPE tail inhibited the regulated secretion of ACTH from AtT20 cells. We also showed that the CPE tail interacted with C-terminus of gamma-adducin, a component of the cytoskeleton that binds and stabilizes F-actin. Overexpression of the C-terminal 38 amino acid of gamma-adducin inhibited the transport of POMC vesicles out of the cell body into the processes of AtT-20 cells. Thus these studies demonstrate that the vesicular CPE cytoplasmic tail plays a novel mechanistic role in anchoring regulated secretory pathway POMC/ACTH and BDNF vesicles to actin via gamma-adducin for movement immediately after budding from the TGN which is actin-based, and subsequently to the microtubule-based motor system for transport along the processes to the plasma membrane for activity-dependent secretion in endocrine cells and neurons. We recently demonstrated that transmembrane CPE is not only associated with large dense core vesicles, but also with glutamate-containing synaptic vesicles in mouse hypothalamus. High K+ stimulated release of glutamate from cultured hypothalamic neurons was absent in CPE-KO mice. Furthermore, electron microscopic analysis of 100 hypothalamic synaptic densities revealed that 40% of the synapses had no docked synaptic vesicles at the presynaptic density in CPE-KO mice in contrast to 15% for the WT mice, implicating that in some neurons, CPE may be involved in synaptic vesicle tethering/docking, possibly mediated by its cytoplasmic tail. Indeed, in vitro GST pulldown assays using both brain and PC12 cell cytosol, GST tagged CPEC10, the CPE cytoplasmic tail, bound Rab27A, Rab3A and Rim1, molecules necessary for synaptic vesicle tethering, but not munc18 required for docking to the synaptic membrane. Furthermore TIRF microscopy showed that expression of GFP-tagged CPE tail in PC12 cells reduced the steady-state level of the total number of dynamically mobile synaptophysin-mRFP synaptic-like vesicles in the sub-plasma membrane area. Taken together these findings have uncovered a new mediator, the CPE cytoplasmic tail, in synaptic vesicle tethering at the plasma membrane in a sub-population of hypothalamic neurons and in PC12 cells, through interaction with Rim 1, Rab27A and Rab3A. The mechanism regulating biogenesis of hormone/neuropeptide containing large dense-core secretory granule (LDCG) to replenish these organelles after stimulus-coupled secretion was investigated. We previously showed that chromogranin A (CgA) plays a critical role in the control of LDCG formation and that the mechanism was through the control of granule protein degradation within the Golgi by regulating the level of a protease inhibitor. We have identified the inhibitor as protease nexin-1 (PN-1) which expression was up-regulated transcriptionally by CgA (or a fragment of CgA). PN-1 prevented granule protein degradation and increased LDCG formation when up-regulated, but when reduced in its expression by PN-1 antisense-RNA, the proteins were degraded and LDCGs were not made. We recently identified a C-terminal fragment of CgA, which we named serpinin that was able to enhance PN-1 transcription and granule biogenesis in 6T3 cells, an endocrine cell line that normally lacks LDCGs. Serpinin was elevated in the medium after high K+ stimulation of AtT20 cells. We further demonstrated that serpinin increases cAMP presumably by binding to a cognate receptor on the plasma membrane to cause signaling to the nucleus to enhance PN-1 mRNA transcription. Thus we have discovered a new CgA-derived peptide, serpinin, which is co-secreted with POMC-derived hormones upon stimulation of pituitary corticotrophs. Serpinin signals replenishment of LDCGs by transcriptionally up-regulating the expression of the protease inhibitor, PN-1 which then stabilizes granule proteins, resulting in increased LDCG biogenesis. Recently, we have investigated the role of CPE in the nervous system in vivo. We showed that CPE KO mice were not able to process pro-CART to CART and therefore lacked this anorexigenic neuropeptide, in the hypothalamus. These animals over-eat, thus providing further evidence linking decrease of this neuropeptide to the cause of obesity. Additionally, in collaboration with the Accili group at Columbia University, it was found that the transcription factor FoxO1 negatively regulates CPE gene expression. Normally insulin binds to insulin receptors in the POMC neurons and that leads to nuclear signaling, nuclear exclusion and inactivation of FoxO1. To model this physiological event, FoxO1 was deleted in the POMC neurons in the arcuate nucleus of the hypothalamus in mice and that resulted in increased CPE levels, increased &#61537;-MSH, an anorexigenic neuropeptide derived from POMC, and reduced food intake without change in energy expenditure. These findings raise the possibility of targeting CPE to develop weight loss medications. Also we showed that the extremely obese CPE KO mice have low bone mineral density, contrary to dogma that obesity is always associated with high bone mineral density. We further concluded that the lack of CART which promotes bone formation, is an important player responsible for poor bone density in these mice. Also we demonstrated deficiencies in several behavioral assays including the Morris water maze and object preference tests indicating a problem with learning and memory in CPE-KO mice. We showed that in 6-14 week old CPE-KO mice, dendritic pruning was inhibited in cortical and hippocampal neurons which would affect synaptogeneis. Additionally electrophysiological measurements showed a defect in the generation of long term potentiation (LTP) in hippocampal slices of these mice. A major cause for this defect was due to the total degeneration of neurons in the CA3 region of the hippocampus. Interestingly, this was evident only in 4 week and older CPE-KO mice. Three week old KO animals were normal, suggesting that CPE is important in maintaining the survival of CA3 neurons which are enriched in this enzyme, after 3 weeks of age. These results have therefore uncovered a critical period between 3 and 4 weeks after birth when the CA3 neurons are highly sensitive to stress such as maternal separation, and that CPE is required to maintain survival of these neurons after 3 weeks of age. Indeed, we showed that when CPE was overexpressed in hippocampal neurons in culture, they were protected from apoptosis after induced oxidative stress using hydrogen peroxide. Thus, CPE has a novel neuroprotective role in adult hippocampal neurons. The CPE-KO mouse is therefore an excellent model for studying the critical period of CA3 neuronal survival and the effects of early maternal separation on infant brain development.